Use of a recombinant pseudorabies virus to analyze motor cortical reorganization after unilateral facial denervation

A unilateral facial nerve injury (n7x) was found to influence the transcallosal spread of the attenuated strain of pseudorabies virus (PRV Bartha) from the affected (left) primary motor cortex (MI) to the contralateral MI of rats. We used Ba-DupLac, a recombinant PRV strain, for the tracing experiments since this virus was demonstrated to exhibit much more restricted transportation kinetics than that of PRV Bartha, and is therefore more suitable for studies of neuronal plasticity. Ba-Duplac injection primarily infected several neurons around the penetration channel, but hardly any transcallosally infected neurons were observed in the contraleral MI. In contrast, after right facial nerve injury, Ba-DupLac was transported from the primarily infected neurons in the left MI to the contralateral side, and resulted in the labeling of several neurons due to a transneuronal infection. These results reveal that a peripheral nerve injury induces changes in the Ba-DupLac infection pattern in the related cortical areas. These findings and the literature data suggest that this phenomenon may be related to the changes in the expression or to the redistribution of cell-adhesion molecules, which are known to facilitate the entrance and/or transmission of PRV into neurons

A szívritmuszavarok és a myocardiális repolarizáció mechanizmusainak vizsgálata; antiaritmiás és proaritmiás gyógyszerhatások elemzése = Study of the mechanism of cardiac arrhythmias and repolarization, antiarrhythmic and proarrhythmic drug action

A kardiovaszkuláris betegségek és azon belül is az életet veszélyeztető kamrai és pitvari aritmiák a fő halálozási okok közé tartoznak a fejlett ipari országokban, de Magyarországon is. Ezzel összhangban, a jelen kutatási projekt is a különböző életet veszélyeztető aritmiák megelőzésének a lehetőségét, és a különböző gyógyszerek antiaritmiás és proaritmiás hatásainak a kutatását tűzte ki célul. Vizsgálataink során megállapítottuk, hogy kísérletes diabetes mellitusban mind kutyán, mind nyúlon az IKs áram és következményesen a repolarizációs rezerv csökkenése következik be amely emberben vélhetően hozzájárulhat e betegségben észlelt hirtelen szívhalál kockázat növekedéséhez. Molekuláris biológiai vizsgálatokban sikerült feltérképeznünk az emberi szívizom különféle ioncsatornáinak denzitását is. A proaritmiás gyógyszerhatások elemzésére újszerű módszert dolgoztunk ki, amelynek gyógyszerbiztonsági klinikai jelentősége és hasznosulása várható. A projekt teljesítése során további új ismereteket szereztünk a Na+/Ca2+ cseremechanizmus (NCX) repolarizációban betöltött szerepét illetően. In vivo kutya kísérletekben vizsgáltuk a peroxynitrit és gap junction csatornák szerepét az ischaemiás prekondicionálásban. Ezek az eredmények várhatóan hozzájárulnak egyrészt a szívizomzat élettani, kórélettani (aritmia mechanizmusok) ismereteinek a gyarapításában, másrészt új és biztonságos antiaritmiás terápiák kifejlesztéséhez. | Cardiovascular diseases, including life threatening ventricular and supraventricular arrhythmias, are the leading causes of mortality in industrialized countries and also in Hungary. In harmony with this, the major goal of the project was to investigate the mechanisms involved in cardiac repolarization and in antiarrhythmic and proarrhythmic drug actions. Representing important findings during the project, we established that in experimental diabetes mellitus the IKs potassium current is down-regulated resulting in the attenuation of repolarization reserve which may contribute to the increased proarrhythmic risk of diabetic patients. Using molecular biological methods we have analyzed the transmembrane ion channel densities of the human heart. To assess proarrhyhtmic drug side effects we developed a novel method which can be expected to contribute to better prediction of proarrhythmic risk in both preclinical and clinical safety pharmacology investigations. During the project, we have gained further insights regarding the role of NCX in the cardiac repolarization process. In in vivo studies we have investigated the possible role of peroxynitrite and gap junctions in ischaemic preconditioning. These results can be expected to help to better understand the physiology and pathophysiology of cardiac muscle, and arrhythmias, and should significantly contribute to the development of safer and more effective antiarrhythmic treatment modalities

The Neurotropic Parasite Toxoplasma Gondii Increases Dopamine Metabolism

The highly prevalent parasite Toxoplasma gondii manipulates its host's behavior. In infected rodents, the behavioral changes increase the likelihood that the parasite will be transmitted back to its definitive cat host, an essential step in completion of the parasite's life cycle. The mechanism(s) responsible for behavioral changes in the host is unknown but two lines of published evidence suggest that the parasite alters neurotransmitter signal transduction: the disruption of the parasite-induced behavioral changes with medications used to treat psychiatric disease (specifically dopamine antagonists) and identification of a tyrosine hydroxylase encoded in the parasite genome. In this study, infection of mammalian dopaminergic cells with T. gondii enhanced the levels of K+-induced release of dopamine several-fold, with a direct correlation between the number of infected cells and the quantity of dopamine released. Immunostaining brain sections of infected mice with dopamine antibody showed intense staining of encysted parasites. Based on these analyses, T. gondii orchestrates a significant increase in dopamine metabolism in neural cells. Tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, was also found in intracellular tissue cysts in brain tissue with antibodies specific for the parasite-encoded tyrosine hydroxylase. These observations provide a mechanism for parasite-induced behavioral changes. The observed effects on dopamine metabolism could also be relevant in interpreting reports of psychobehavioral changes in toxoplasmosis-infected humans

Behavioral Abnormalities in a Mouse Model of Chronic Toxoplasmosis Are Associated with MAG1 Antibody Levels and Cyst Burden.

There is marked variation in the human response to Toxoplasma gondii infection. Epidemiological studies indicate associations between strain virulence and severity of toxoplasmosis. Animal studies on the pathogenic effect of chronic infection focused on relatively avirulent strains (e.g. type II) because they can easily establish latent infections in mice, defined by the presence of bradyzoite-containing cysts. To provide insight into virulent strain-related severity of human toxoplasmosis, we established a chronic model of the virulent type I strain using outbred mice. We found that type I-exposed mice displayed variable outcomes ranging from aborted to severe infections. According to antibody profiles, we found that most of mice generated antibodies against T. gondii organism but varied greatly in the production of antibodies against matrix antigen MAG1. There was a strong correlation between MAG1 antibody level and brain cyst burden in chronically infected mice (r = 0.82, p = 0.0021). We found that mice with high MAG1 antibody level displayed lower weight, behavioral changes, altered levels of gene expression and immune activation. The most striking change in behavior we discovered was a blunted response to amphetamine-trigged locomotor activity. The extent of most changes was directly correlated with levels of MAG1 antibody. These changes were not found in mice with less cyst burden or mice that were acutely but not chronically infected. Our finding highlights the critical role of cyst burden in a range of disease severity during chronic infection, the predictive value of MAG1 antibody level to brain cyst burden and to changes in behavior or other pathology in chronically infected mice. Our finding may have important implications for understanding the heterogeneous effects of T. gondii infections in human

Spatiotemporal changes of the herpes simplex virus entry receptor nectin-1 in murine brain during postnatal development

Herpes simplex virus (HSV) is known to replicate within the limbic system and to alter behavior in both humans and experimental animals. However, the reason why the virus selectively damages this anatomical, developmental, and functional neural unit remains a mystery. Nor is it known why herpes simplex encephalitis fails to respect these neuroanatomical boundaries in newborns. In the present study, the authors determined the spatiotemporal changes in the distribution of the major neural entry receptor for HSV (nectin-1) in postnatal mouse and rat brains. Discrete nectin-1 immunopositivity was observed in regions susceptible to HSV infection in specific developmental phases of central nervous system. The authors also describe nectin-1-related pathways controlling neuronal cell migration/brain morphogenesis, the disruption of which might lead to the emergence of mental disorders with a rapid cognitive decline

Dopamine in tissue cysts of <i>T. gondii</i> in brain tissue sections.

<p>(A) Dopamine was detected in brain tissue sections of chronically infected Swiss Webster mice by immunohistochemical staining with anti-dopamine antibody and horseradish peroxidase. Tissue cysts containing hundreds of bradyzoites are visible as brown circular structures (arrowheads) in infected brains. The bottom right panel is a control lacking anti-dopamine antibody. All black bars are 10 µm long. (B) Localization by indirect immunofluorescence of brain sections stained with anti-dopamine antibody (green), DAPI (blue), and TRITC-lectin (red). Three sections are shown from different regions of the brain in the top, middle and bottom rows of panels with the negative control (no primary antibody) in the bottom row. In each series all three channels are illuminated (left), the anti-dopamine and lectin channels are illuminated (center), and only the anti-dopamine channel is illuminated (right). The DAPI identifies neural cells and the individual bradyzoites within the tissue cyst and the lectin stains the surface of the cyst. The dopamine staining appeared specific (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023866#pone-0023866-g002" target="_blank">Fig. 2</a>) as the antibody stained neurons in the striatum, amygdala and hippocampus. (C) A 3D projection of a Z-stack reconstruction of serial images of a tissue cyst within a brain section stained with anti-dopamine antibody and lectin as described in B. Control without the primary anti-dopamine antibody is shown in the right panel.</p

Specificity of dopamine <i>staining T. gondii</i> tissue cysts.

<p>(A) Histochemical (glyoxylic acid) staining of dopamine in brain sections from chronically-infected mice detected by fluorescence. Glyoxylate reacts with dopamine to fluoresce blue-white <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023866#pone.0023866-Lent1" target="_blank">[17]</a>. Cells containing <i>T. gondii</i> cysts in brain tissue exhibited blue-white fluorescence. The tissue cysts stained darkly, similar to mouse cell nuclei, presumably due the high density of bradyzoites. (B) Brain tissue sections from chronically-infected mice were stained with indirect fluorescein staining as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023866#pone-0023866-g001" target="_blank">Fig. 1</a> except the anti-dopamine primary antibody was incubated in the presence of 50 µg/ml dopamine (top) and 50 µg/ml serotonin (botto). From left to right: bright field, fluorescein only channel (green), fluorescein and lectin-TRITC (green and red channels, respectively), and both channels plus bright field. Serotonin did not compete for dopamine staining.</p

Dopamine enzyme tyrosine hydroxylase in intracellular <i>T. gondii</i>.

<p>(A) Immunohistochemical localization of tyrosine hydroxylase (TH) in brain sections of chronically-infected mice with commercial antibody and horseradish peroxidase labelling. Tissue cysts are visible as brown circular structures (left, four cysts, and right, single cyst, highlighted with arrowheads). (B) TH in intracellular parasites <i>in vitro</i>. Alkaline-induce parasite cultures were probed with anti-tyrosine hydroxylase antibody (green), RFP-GRASP (red), and DAPI (blue) shown separately and as a composite image. Scale bars on all images are 10 µM.</p